Poxviruses include a large family of DNA viruses capable of infecting and causing disease in humans. While the most notorious member variola, the causative agent of smallpox, was eradicated from natural infection, there are still concerns about a clandestine release during a biological attack. In addition, monkeypox and other members of the family have raised concern about epizootic infections that are capable of causing epidemic. Poxviruses produce two infectious forms, intracellular mature virus (IMV) and extracellular virus (EV). IMV make up the majority of progeny virions. EV are formed by the intracellular envelopment of IMV and are required for cell-to-cell spread and systemic infection. Only 8 viral proteins are known to be unique to the EV form. The long-term goal of this project is to understand the molecular mechanisms employed by orthopoxoviruses to envelope, transport, and release infectious EV. The immediate goal of this application is to understand how interactions with, and amongst the three major EV glycoproteins facilitate proper protein content in the EV envelop and how this effects systemic infection. Our specific aims are; 1) Fine mapping of specific residues required for EV protein interaction, 2) To determine the hierarchy and spatial relationship between the EV protein interactions. 3) To determine the relationship between EV protein interaction EV envelope protein composition, infectious EV production, and pathogenesis in vivo. The conclusion of this study will provide information about the interaction between the three major orthopoxvirus glycoproteins (A33, A34, and B5) at the molecular, cellular and organismal levels. The results obtained will provide greater insight into the molecular mechanism poxviruses use to produce infectious EV and spread cell-to-cell in its host. In addition they will inform intelligent design decision when constructing recombinant poxvirus vectors for both vaccines and oncolytic platforms.